An NMR approach to study functional GPCRs obtained by directed evolution

研究通过定向进化获得的功能性 GPCR 的 NMR 方法

• 批准号: 466160427
• 负责人: Professor Dr. Franz Hagn
• 金额: --
• 依托单位: Professur für Strukturelle Membranbiochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/466160427?language=en
• 关键词: NMR; approach; study; functional; GPCRs

G-protein coupled receptors (GPCRs) are essential components of the signal transduction pathways between cells and thus are targets of around 60% of all currently marketed drugs. Despite a growing number of crystal and EM structures, the mechanism of GPCR-mediated signaling is still not sufficiently well understood as activation and switching properties of GPCRs cannot be fully explained by static snapshots. In this project, we will characterize the structural and dynamical changes in a GPCR upon binding to an antagonist, agonist or G-protein using solution-state NMR spectroscopy as a main tool. In order to be able to study such a receptor system by NMR at high resolution and quality, high-level stable isotope labeling schemes (esp. high deuteration and selective methyl labeling) are essential. Bacterial expression systems (such as E. coli) are best suited for these special requirements. However, until recently, the production yields of GPCRs in E. coli have been very low. This unfavorable situation was fundamentally changed by the application of directed evolution methods to GPCRs, rendering high resolution NMR studies on this essential membrane protein class feasible. In the proposed study, we will use the well-characterized neurotensin receptor subtype-1 (NTR1) as a model system to develop a strategy to investigate a stabilized GPCRs by solution-state NMR. Furthermore, we will apply a rational NMR-based approach to rescue the biased functionality of this mutated GPCR. Preliminary results provide a promising picture that relevant solution state NMR investigations can be conducted on the mechanism of NTR1 activation by small molecule ligands as well as a G-protein using a functional and stabilized receptor variant. These experiments will contribute to a better understanding of the allosteric switching properties of a GPCR as well as provide new approaches for the structural investigation of GPCRs by solution state NMR.

G 蛋白偶联受体 (GPCR) 是细胞间信号转导途径的重要组成部分，因此是目前上市药物中约 60% 的靶标。尽管晶体和 EM 结构的数量不断增加，但 GPCR 介导的信号传导机制仍然没有得到充分理解，因为静态快照无法完全解释 GPCR 的激活和转换特性。在这个项目中，我们将使用溶液态核磁共振波谱作为主要工具来表征 GPCR 与拮抗剂、激动剂或 G 蛋白结合后的结构和动力学变化。为了能够通过核磁共振以高分辨率和高质量研究这样的受体系统，高水平的稳定同位素标记方案（特别是高氘化和选择性甲基标记）是必不可少的。细菌表达系统（例如大肠杆菌）最适合这些特殊要求。然而，直到最近，GPCR 在大肠杆菌中的产量一直非常低。 GPCR 定向进化方法的应用从根本上改变了这种不利局面，使得对这一重要膜蛋白类别的高分辨率 NMR 研究变得可行。在拟议的研究中，我们将使用已充分表征的神经降压素受体亚型 1 (NTR1) 作为模型系统，制定通过溶液态 NMR 研究稳定 GPCR 的策略。此外，我们将应用一种基于 NMR 的合理方法来挽救这种突变 GPCR 的有偏差的功能。初步结果提供了一个有希望的前景，即可以使用功能性和稳定的受体变体，对小分子配体以及 G 蛋白激活 NTR1 的机制进行相关的溶液态 NMR 研究。这些实验将有助于更好地理解 GPCR 的变构转换特性，并为通过溶液态 NMR 进行 GPCR 结构研究提供新方法。